Interferons (IFNs) are members of a group of intercellular messenger proteins known as cytokines. .gamma.-IFN, also known as type II IFN, is produced in T-cells and natural killer cells. Biosynthesis of .gamma.-IFN is stimulated by antigens to which the organism has been sensitized. IFNs' immunomodulatory and anti-inflammatory actions are part of the body's natural defense against viruses and cancers. They exert these defenses by affecting the function of the immune system and by direct action on pathogens and cancer cells. IFNs mediate these multiple effects in part by inducing the synthesis of many cellular proteins. Some IFN induced (IFI) proteins are preferentially induced by .gamma.-IFN.
The various IFI proteins possess anti-cancer, antiviral and immunomodulatory functions. For example, IFI proteins are known to inhibit viral functions such as cell penetration, uncoating, RNA and protein synthesis, assembly and release (cf Hardman J. G. et al. (1996) The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y. pp 1211-1215). Furthermore, induction of major histocompatibility complex (MHC) proteins by .gamma.-IFN can enhance an immune response enhancement.
The human macrophage cell line, RAW 264.7, responds to stimulation with .gamma.-IFN plus lipopolysaccharide (LPS) by the development of cytolytic activity. Two closely homologous genes (76% identity in amino acid sequence) were found to be induced by .gamma.-IFN stimulation of RAW 264.7 cells, but not induced in the non-tumoricidal line WEHI-3 (Wynn T. A. et al. (1991) J. Immunol. 147: 4384-4392). The protein products of these genes were found to selectively bind guanine mono, di, and tri phosphate (GMP, GDP, and GTP) and were therefore named guanylate-binding proteins 1 and 2 (GBP-1 and GBP-2; Cheng Y. E. et al. (1991) Mol. Cell. Biol. 11: 4717-4725). Sequence analysis revealed that the GBPs contain two of three highly conserved guanine-nucleotide-binding motifs GXXXXGKS(T) and DXXG, but lack the third, N(T)KXD. Despite the lack of this third binding motif, which was thought to be important for guanine specificity, GBPs interact with GTP but not with ATP or other nucleotides. Experimental evidence revealed that human GBP-1 is a GTPase, hydrolyzing GTP to GMP (Schwemmle M. et al. (1994) J. Biol. Chem. 269: 11299-11305). GDP does not appear to be a major substrate, inhibitor, or product of GBP-1 (Schwemmle et al., supra). Recently, human GBP-2 was found to hydrolyze GTP to GDP (Neun R. et al. (1996) FEBS Lett. 390: 69-72). Thus, although human GBP-1 and GBP-2 appear to have similar biological activities, they show differences in product specificity.
Indirect immunofluorescence analysis of both .gamma.-IFN treated human cells and transfected mouse cells, using antibodies specific for human GBP-1, showed a staining pattern that suggested that GBP-1 was associated with the inner cell membrane (Schwemrle et al., supra). Additionally, in vitro assays showed that an isoprenylation motif, CaaX, at the C terminus of human GBP-1 does indeed function as a isoprenylation signal, allowing the addition of a 20-carbon molecule that serves to anchor the protein to a membrane (Schwemmle et al., supra). GBP-2 also contains the CaaX motif, thus both GBPs appear to be membrane-associated. A rat GBP was found to be prenylated in vivo (Vestal D. J. et al. (1996) Biochem. Biophys. Res. Commun. 224: 528-534). The rat GBP was found to be induced by both .gamma.-IFN and LPS in cultured macrophages and microglia (Vestal et al. supra). LPS induction of human GBPs has not been reported. The expression patterns of human GBP-1 and GBP-2 are distinct, but both appear to be restricted to .gamma.-IFN induced macrophages and fibroblasts. Although the specific function of GBPs is not known, expression pattern and aspects of their amino acid sequence suggest that GBPs are important agents in the development of tumoricidal macrophages (supra). GBPs have also been characterized as potential intracellular mediators of .gamma.-IFN induced antiviral effects (Chen et al., supra).
The discovery of polynucleotides encoding human guanylate-binding proteins produced by immune system cells, and the molecules themselves, presents the opportunity to investigate .gamma.-IFN mediated anti-viral, anti-inflammatory, and anti-cancer activities. Discovery of molecules related to GBPs satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the diagnosis and treatment of cancer, viral infections, inflammation, or conditions associated with impaired immunity.